טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentHofshi Anat
SubjectA Combined Gene and Cell Therapy Approach for the
Treatment of Cardiac Arrhythmias
DepartmentDepartment of Medicine
Supervisor Professor Lior Gepstein
Full Thesis textFull thesis text - English Version


Abstract

Impaired myocardial conduction may underlie both bradyarrhythmias and reentrant tachyarrhythmias. Arryhthmic disorders are a significant cause of morbidity and mortality in the western world. Conduction restoration may therefore represent an important antiarrhythmic target.

This work presents a novel strategy for conduction repair utilizing genetically engineered cells designed to form biological “conducting-cables”. An in-vitro model of conduction block was established using spatially-separated, non-synchronized, human embryonic stem cell-derived cardiomyocyte-clusters. Different experiments were performed to evaluate the ability of genetically-engineered HEK293 cells, expressing the Nav1.5 sodium channel, to couple with cultured cardiomyocytes and to synchronize their electrical activity. Further studies focused on improving the conduction properties of the “biological-cables” by co-expressing the potassium Kir2.1 channel in the Nav1.5-HEK293 cells. The Kir2.1 channel was expected to induce hyperpolarization of the engineered cells’ resting membrane potential (RMP) which is required for increasing the number of available sodium channels.

Connexin-43 immunostaining and Calcein dye-transfer experiments confirmed formation of functional gap-junction between the engineered cells and neighboring cardiomyocytes. Multielectrode array (MEA) and intracellular recordings were performed to assess the engineered cells' ability to restore and synchronize conduction in the co-cultures. Synchronization was defined by establishment of fixed local activation time differences between the cardiomyocyte-clusters and convergence of their spontaneous activation cycle-lengths. Importantly, Nav1.5-HEK293 cells facilitated synchronization by up to 1050μm, while non-transfected control cells could induce synchronization only in distances up to 300μm.

Next, introduction of Kir2.1 channel into the Nav1.5-HEK293 cells resulted in RMP hyperpolarization. Interestingly, the engineered cells expressing both channels transformed into excitable cells and following depolarization could fire “all-or-none” action potentials. Moreover, the Nav1.5-Kir2.1-HEK293 cells also enabled synchronization between clusters separated up to 5.5mm (the longest distance studied due to MEA plate size limitation ), which is 5.5 fold more distant than the maximal coupling distance facilitated by the cells expressing only the sodium channels. The synchronization occurred within hours after establishing the co-culture and in 100% of the co-cultures examined. The conduction velocity along the “conducting-cables” improved considerably from 0.403±0.244cm/sec to 4.72±1.27cm/sec. Furthermore, Kir2.1 channel co-expression in the Nav1.5-HEK293 cells enabled a uniform and homogenous conduction.

In summary, genetically-engineered cells, transfected to express Na+ and K+ channels can form “biological conducting cables” coupling and synchronizing spatially-separated cardiomyocytes. This novel cell and gene therapy approach may enable development of strategies targeting different brady - and tachy-arrhythmias.